The overall goal of our laboratory's research is to develop cellular engineering strategies for the cure of diabetes by better understanding the normal processes of differentiation that occur in the embryonic pancreas, and that lead to normal differentiation of pancreatic beta-cells from undifferentiated pancreatic precursor cells in the embryonic foregut. We have shown that the embryonic mouse pancreatic epithelium, shortly after evagination from the foregut, has the capacity to form all three components of the pancreas (islets, ducts, and acini), and that the extracellular milieu during development is critical to determining the specific type of pancreatic cells that will form. We now, in this grant proposal, wish to expand our knowledge about these early epithelial cells, and the mechanism by which pro-endocrine differentiation (and beta-cell differentiation in particular) is normally induced by the epithelium milieu. Toward this goal, we have developed three specific aims. First, we will attempt to create antibodies specific for this epithelium that are directed against cell surface markers, to allow for sorting for selecting pancreatic embryonic epithelial-like cells from large populations of stem cells described in other projects within this overall proposal. The creation of such antibodies will be facilitated by making immortalized cell lines from representative embryonic cells, to facilitate screening of potential antibody supernatants. Second, the unique gene expression pattern of these early pancreatic epithelial cells will be better characterized using comparative cDNA array analyses as well as a suppressive subtractive hybridization approach. Given the importance of the phenotype of these epithelial cells, this second specific aim will allow critical phenotypic markers to be determined and compared with candidate derived from all four of the projects in this overall proposal. Third, the pancreatic lineage selection and differentiation induced by critical cytokine pathway (TGF- beta superfamily signaling) and a critical extracellular matrix pathway (laminin) will be studied mechanistically toward optimizing growth conditions to induce pancreatic embryonic epithelial-like cells to become endocrine cells. Together, these specific aims should work synergistically with the other projects in this overall grant proposal to greatly enhance our understanding of possible avenues for engineering of stem cells to become pancreatic endocrine/beta-cells.